Device, system, and method for selecting a target analyte or fluid

ABSTRACT

This disclosure is directed to a device and a system for aspirating and dispensing a target analyte, target material, or fluid. A picker may aspirate and dispense the desired material by introducing a pressure gradient. The picker may include a hydraulic fluid to hydraulically couple at least two components, such as a moveable pump component and a cannula.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation of application Ser. No. 14/643,891,filed Mar. 10, 2015, which is a continuation-in-part of application Ser.No. 14/248,510, filed Apr. 9, 2014, which claims the benefit ofProvisional Application No. 61/810,834, filed Apr. 11, 2013, andProvisional Application No. 61/922,931, filed Jan. 2, 2014.

TECHNICAL FIELD

This disclosure relates generally to micromanipulation of a targetanalyte, though more specifically, to picking and isolating the targetanalyte.

BACKGROUND

Suspensions often include materials of interest that are difficult todetect, extract and isolate for analysis. For instance, whole blood is asuspension of materials in a fluid. The materials include billions ofred and white blood cells and platelets in a proteinaceous fluid calledplasma. Whole blood is routinely examined for the presence of abnormalorganisms or cells, such as fetal cells, endothelial cells, epithelialcells, parasites, bacteria, and inflammatory cells, and viruses,including HIV, cytomegalovirus, hepatitis C virus, and Epstein-Barrvirus, and nucleic acids. Currently, practitioners, researchers, andthose working with blood samples try to separate, isolate, and extractcertain components of a peripheral blood sample for examination. Typicaltechniques used to analyze a blood sample include the steps of smearinga film of blood on a slide and staining the film in a way that enablescertain components to be examined by bright field microscopy.

On the other hand, materials of interest composed of particles thatoccur in very low numbers are especially difficult if not impossible todetect and analyze using many existing techniques. Consider, forinstance, circulating tumor cells (“CTCs”), which are cancer cells thathave detached from a tumor, circulate in the bloodstream, and may beregarded as seeds for subsequent growth of additional tumors (i.e.,metastasis) in different tissues. The ability to accurately detect andanalyze CTCs is of particular interest to oncologists and cancerresearchers, but CTCs occur in very low numbers in peripheral wholeblood samples. For instance, a 7.5 ml sample of peripheral whole bloodthat contains as few as 3 CTCs is considered clinically relevant in thediagnosis and treatment of a cancer patient. However, detecting even 1CTC in a 7.5 ml blood sample may be clinically relevant and isequivalent to detecting 1 CTC in a background of about 50 billion redand white blood cells. Using existing techniques to find, isolate andextract as few as 3 CTCs of a whole blood sample is extremely timeconsuming, costly and is extremely difficult to accomplish.

As a result, practitioners, researchers, and those working withsuspensions continue to seek systems and methods to more efficiently andaccurately detect, isolate and extract target materials of a suspension.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show examples of a picker.

FIG. 2A-2D show an example picker.

FIG. 3A shows an example picker.

FIG. 3B shows an example picker.

FIGS. 4A-4C show an example picker.

FIG. 5 shows an example cannula with a fluorescent tip.

FIGS. 6A-6B show an example fluorescent picker tip.

FIGS. 7A-7E show example picker tips.

FIGS. 8A-8B show an example picking system.

DETAILED DESCRIPTION

This disclosure is directed to a device and a system for aspirating anddispensing a target analyte, target material, or fluid. A picker mayaspirate and dispense the desired material by introducing a pressuregradient. The picker may include a hydraulic fluid to hydraulicallycouple at least two components, such as a moveable pump component and acannula.

Picker and Picking System

FIG. 1A shows an example picker 100. The picker 100 includes a main body102, a back end 104, and a tip 106. The picker 100 may be solid or maybe a hollow tube having an inner chamber for holding a liquid, targetanalyte, or any other appropriate material. When the picker 100 is ahollow tube, the picker 100 may also include a liquid 108 within theinner chamber of the picker 100, where the liquid may be a solution, abuffer, a ferrofluid, or the like. The picker 100 may be used tomanipulate a target analyte. The target analyte may be manipulated, suchas by moving, removal, or isolation, when the specific target analyte isin a vessel, such as a tube or a well, or on a slide. The target analytecan be isolated through the introduction of a force, thereby attractingor pulling the target analyte. The tip 106 engages the target analytefor moving, removal, or isolation. The force may be created with suctionor a pressure gradient, such as a vacuum. The back end 104 may beconnected to a pump 110, such as a vacuum pump, a lead screw, or a handpump with a wheel, to aid in providing the force for moving, removal, orisolation. The picker 100 may also include a light source 112, such asan LED, to illuminate an area in which the target analyte may bepresent. The light source 112 may be located anywhere along the mainbody 102, including the back end 104 and the tip 106. When the lightsource 112 is located at the back end 104, the main body 102 may becomposed of a material capable of propagating or transmitting a lightsignal produced by the light source 112, such that the light signalexits at the tip 106 to illuminate the desired area. The light source112 may be connected to a power supply (not shown), such as a battery,to supply current or power.

FIG. 1B shows an example picker 114. The picker 114 is similar to thepicker 100, except that picker 114 includes a permanent magnet 116, suchas a donut-shaped magnet. The permanent magnet 116 generates a magneticfield for attracting a particle of a target analyte-particle complex, atarget analyte having been previously conjugated with the particle toform the target analyte-particle complex. The picker 114 may alsoinclude a magnetizable material to extend or transmit the magnetic fieldproduced by the magnet. The permanent magnet 116 may be located at thetip 106 or at or near the back end 104. The permanent magnet 116 may beremovable. Alternatively, the fluid, such as a ferrofluid, within thepicker 114 may be used to generate the magnetic field or magneticgradient.

FIG. 1C shows an example picker 120. The picker 120 is similar to thepicker 100, except that picker 120 includes an electromagnet. Theelectromagnet includes a power source 122, a first lead 126, a secondlead 128, and a coil 124. The power source 122 may be, but is notlimited to, a battery, a DC supply, or an AC supply. The electromagnetgenerates a magnetic field for attracting a particle of a targetanalyte-particle complex, a target analyte having been previouslyconjugated with the particle to form the target analyte-particlecomplex. The picker 120 may be composed of a magnetizable material toextend or transmit the magnetic field produced by the magnet. The firstlead 126, the second 128, and the coil 124 may be located outside of awall of the picker 120, may be embedded with the wall of the picker 120,or may be located inside of the picker 120. The picker 120 may alsoinclude a light source 130, such as an LED, to illuminate an area inwhich the target analyte may be present. The light source 130 may belocated anywhere along the main body 102, including the back end 104 andthe tip 106. When the light source 130 is located at the back end 104,the main body 102 may be composed of a material capable of propagatingor transmitting a light signal produced by the light source 130, suchthat the light signal exits at the tip 106 to illuminate the desiredarea. The light source 130 may be connected to a power supply (notshown), such as a battery, to supply current or power.

FIG. 1D shows an example picker 140. The picker 140 includes aretractable shaft 142, the retractable shaft 142 being thinner than themain body 102 and being extendable from the tip 106. The retractableshaft 142 can be located within the main body 102, can be extended outof the tip 106 to engage a target analyte, and can be retracted into themain body 102. When the target analyte attaches to the retractable shaft142, the target analyte can be drawn into the main body 102. Theretractable shaft 142 may include an engagement portion 152, a stopper144, a grip 150, and a rod 148. The engagement portion 152 may beextended out of the tip 106 to engage the target analyte. The stopper144 may be sized to fit within the main body 102, but be larger than thetip 106 or a taper from the main body 102 to the tip 106, therebypreventing the retractable shaft 142 from extending too far from the tip106. The grip 150 may allow for engagement of the retractable shaft 142,so as to properly move the retractable shaft 142. The rod 148 mayconnect the stopper 144 or the engagement portion 146 to the grip 140.The retractable shaft 142 may also be made magnetizable by including amagnet 146 disposed on or within the retractable shaft 142. The magneticfield or magnetic gradient may be removed or deactivated, such as byremoving the magnet 146 or turning off an electromagnet. The targetanalyte-particle complex is no longer attracted and held to theretractable shaft 142 causing the target analyte-particle complex toremain within the liquid in the main body 102. The picker 140 may alsoinclude a light source 130, such as an LED, to illuminate an area inwhich the target analyte may be present. The light source 130 may belocated anywhere along the main body 102, including the back end 104 andthe tip 106. When the light source 130 is located at the back end 104,the main body 102 may be composed of a material capable of propagatingor transmitting a light signal produced by the light source 130, suchthat the light signal exits at the tip 106 to illuminate the desiredarea. The light source 130 may be connected to a power supply (notshown), such as a battery, to supply current or power.

Alternatively, the retractable shaft 142 may be magnetized by anelectromagnet, such as a coil wrapped around a segment of or the entireretractable shaft 142. Alternatively, the picker 140 may include a pump(not shown), such as a vacuum pump, a lead screw, or a hand pump with awheel, to aid in providing the force for moving, removal, or isolation.

FIG. 2A shows an example picker 200. FIG. 2B shows a cross-sectionalview of the example picker 200 taken along the line I-I. The picker 200includes a piston 202, a pump block 204, and a cannula 208. The picker200 may also include a fitting 206 with a first side 218 and a secondside 220. The piston 202 includes a first end 210 and a second end 212.The cannula 208 includes an adapter 214 and a tube end 216, the tube end216 including a picker tip 224. The first side 218 of the fitting 206mates with the pump block 204, such as by a press-fit, detents, notches,complementary threads, or the like. A seal 222 may be formed between thefirst side 218 of the fitting 206 or the adapter 214 of the cannula 208and the pump block 204, such as by an O-ring, grease, silicone grease,or the like, to close the picker 200. The adapter 214 of the cannula 208may mate with the second side 220 of the fitting 206 or the pump block204, such as by a press-fit, detents, notches, complementary threads, orthe like. In other words, the cannula 208, without the inclusion of thefitting 206, may be connected directly to the pump block 204.

The piston 202 may be any appropriate length. The second end 212 of thepiston 202 may be located within the pump block 204, within the fitting206, within the adapter 214 of the cannula 208, or within the tube end216 of the cannula 208. The second end 212 of the piston 210 may extendthrough the seal 222. The first end 210 of the piston 202 may be locatedwithin the pump block 204 or may extend out of a side of the pump block204 opposite the side of the pump block 204 that is connected, whetherdirectly or indirectly, to the cannula 208. The piston 202 and thecannula 208 may substantially share a central axis. The positioning ofthe piston 202 relative to the cannula 208 reduces or eliminates deadvolume.

The pump block 204 at least partially houses the piston 202 and allowsfor translation of the piston 202 relative to the pump block 204. Movingthe piston 202, such as a lead screw or rod, upwards within the pumpblock 204 may create a negative pressure at the tube end 216 so as todraw a target analyte or fluid from the suspension into the cannula 208or may create a positive pressure to expel a target analyte or fluidlocated within the cannula 208 from the tube end 216. The piston 202 maybe connected to a motor or an actuator to drive the piston 202 up anddown, thereby creating the desired pressure differential. The pump block204 may include a complementary mating feature, such as threads or abore, to accept and mate with the piston 202. When the piston 202 andthe pump block 204 include complementary threads, the piston 202 may berotated to cause the desired translation. A full rotation of the piston202 may include any number of steps, including 1-10,000 steps. Thosesteps may then include any number of micro-steps, including 1-10,000micro-steps. Each step or micro-step may draw in a volume approximatelyequal to or less than 1 picoliter, 10 picoliters, 100 picoliters, 1nanoliter, 1 microliter, or 1 milliliter. A piezo-electric pump (notshown) may also be placed in series with the piston 202, such as on thesecond 212 of the piston 202, thereby allowing for even smaller volumesto be processed. The piezo-electric pump (not shown) may be connected toa voltage source (not shown) to cause the deformation required togenerate the desired negative or positive pressure.

The piston 202 and the cannula 208 may be hydraulically coupled, suchthat the volume from the tube end 216 of the cannula 208 to the seal 222is filled with a hydraulic fluid 226. The volume from the tube end 216of the cannula 208 to the seal 222 filled with the hydraulic fluid 226,which may also be referred to as a pump volume, may be contained in arigid structure, rather than a flexible structure, to maintain thehydraulic coupling efficiency. The hydraulic fluid 226 may beincompressible or have low compressibility. The hydraulic fluid 226 maybe a solution, an oil, a liquid metal, a buffer, water, or the like.Hydraulically coupling the piston 202 and the cannula 208 providesbetter small volume control (i.e. full piston travel draws/expels 1-50μL) than a non-hydraulically coupled picker (i.e. filled with air). Thehydraulic fluid 226 may include a fluid plug, such that two volumes ofthe hydraulic fluid 226 are separated by a volume of air or a differentliquid. The pump volume, which is constant, satisfies the conditiongiven by:

V _(P) =V _(HF) +V _(MPC) +V _(PM) +V _(Air),

where V_(P) represents the pump volume, V_(HF) represents the volume ofthe hydraulic fluid, V_(MPC) represents the volume of the portion of theat least one movable pump component within the pump volume, where V_(PM)represents the volume of any picked or aspirated material, and whereV_(Air) represents the volume of air within the pump volume. In thisinstance, the at least one moveable pump component is the piston 202.Because the pump volume is constant and the piston 202 may besubstantially cylindrical, the volume of the piston 202 satisfies theconditions given by:

V _(MPC) =πr ² h,

where V_(MPC) represents the at least one moveable pump component (i.e.the piston 202), r represents the radius of the second end 212 of thepiston 202, h represents the amount of the piston 202 within the pumpvolume. Alternatively, the piston 202 may be substantially square,rectangular, triangular, pentagonal, or any other appropriate polygonalshape. Accordingly, the volume of the piston 202 would satisfy theequations for the volumes of the respective shapes.

FIG. 2C shows the picker 200 with the piston 202 having been driventowards the picker tip 224. The distance (d) traveled by the piston 202may be used to calculate the new volume of the piston 202 within thepump volume based on the shape of the piston 202. The distance (d) maybe positive (piston 202 moves towards picker tip 224; and, accordingly,creates a positive pressure gradient) or negative (piston 202 moves awayfrom picker tip 224; and, accordingly, creates a negative pressuregradient). The new volume of the piston 202 therefore satisfies theconditions given by:

V _(MPC) =πr ²(h+d),

where V_(MPC) represents the at least one moveable pump component (i.e.the piston 202), r represents the radius of the second end 212 of thepiston 202, h represents the amount of the piston 202 within the pumpvolume, and d represents the distance traveled by the piston 202 withinthe pump volume. With respect to the pump volume equation discussedabove (V_(P)=V_(HF)+V_(MPC)+V_(PM)+V_(Air)), the pump volume remainsconstant because the difference between the first and second volumesoccupied by the at least one moveable component (i.e. piston 202) isequal to the amount of the hydraulic fluid 226, picked or aspiratedmaterial, and/or air displaced from the pump volume.

FIG. 2D shows the picker 200 with the piston 202 having been driven awaythe picker tip 224 and drawing in a target material 230 and air 232.Though FIGS. 2B-2D depict the piston 202 being driven towards and thenaway from the picker tip 224 to draw in the target material 230, andconsequently some air 232, the piston 202 may be driven in anyappropriate manner and in appropriate amount to create the desiredpressure gradient. It should be further noted that the air 232 may notbe drawn in when drawing in the target material 230. The target material230 may include, but is not limited to, biological matter (i.e. cells,tissue, biological fluid, etc.) or other fluids (i.e. phosphate bufferedsaline, enzymatic fluids, adherent solutions, water, etc.).

The picker 200 may introduce a magnetic gradient as well, such as by apermanent magnet or an electromagnet, as shown in FIGS. 1B and 1C,respectively, whereby the cannula 208, the hydraulic fluid 226, or thetube end 216 is magnetizable so as to propagate the magnetic gradient.The permanent magnet may be located along the tube end of the cannula,on the piston, or anywhere on the picker tip. When a ferrofluid primesthe cannula, the permanent magnet may be located near the adapter. Theelectromagnet includes a coil, a first lead, a second lead, and a powersupply, such as a battery. The coil wraps around the tube end of thecannula or the picker tip. A first end of the first lead is connected tothe power supply and a second end of the first lead is connected to afirst end of the coil. A first end of the second lead is connected tothe power supply and a second end of the second lead is connected to asecond end of the coil. The power supply is disposed outside of the pumpblock.

FIG. 3A shows a picker 300. The picker 300 is similar to the picker 200except that the picker 300 includes a light source 302. The light source302 produces a light signal that is propagated or transmitted by thecannula 208 or picker tip inserted into, over, or in-line with thecannula 208. The cannula 208 or the picker tip may be composed of amaterial capable of propagating or transmitting the light signalproduced by the light source 302, such that the light signal exits atthe tube end 216 of the cannula 208 or the end of the picker tipfurthest away from the pump block 204 to illuminate the desired areaand/or stimulate a fluorescent probe bound to a target analyte. When thelight source 302, such as an LED, originates at a location other thanthe tube end 216 of the cannula 208 or the end of the picker tipfurthest away from the pump block 204, a cable 304, such as a fiberoptic cable, may transmit the light signal to the tube end 216 of thecannula 208 or the end of the picker tip furthest away from the pumpblock 204 for illumination and/or stimulation purposes. The light source302 may provide epi-, transmitted, or oblique illumination. The lightsource 302 may be connected to a power supply (not shown), such as abattery, to supply current or power. Alternatively, the light source 302may be between the top of the adapter of the cannula 208 and the fitting206. Alternatively, at least one light source 302 may be embedded in thetube end of the cannula 208. Alternatively, the light source 302 may belocated on the adapter 214 or the pump block 204.

FIG. 3B shows a picker 310. The picker 310 is similar to the picker 200except that the picker 310 includes a port 312. The port 244 may extendthrough the second end of the pump block 204 and either the first end218 of the fitting 206 or the adapter 214 of the cannula 208. The port312 may be connected to a loader 314, such as a piezoelectric pump, tofill the pump volume. The loader 314 may be connected to a reservoir 316with a tube 318. The tube 318 may be rigid or flexible. Alternatively,the loader 314 may be interchanged with a pressure sensor (not shown) tomeasure the pressure within the pump volume to make sure the picker 310does not clog.

FIG. 4A shows a picker 400. FIGS. 4B and 4C show cross-section views ofthe picker 400 taken along the line II-II. The picker 400 includes apump block 402 with a diaphragm 404. The picker 400 also includes thecannula 208, as discussed above, and the seal 222, as discussed above.Further, the seal 222 may be formed between the adapter 214 of thecannula 208 or the fitting (not shown) and the pump block 402, such asby an O-ring or silicone grease, to close the picker 400. The adapter214 of the cannula 208 may mate with the fitting (not shown) or the pumpblock 402, such as by a press-fit, detents, notches, complementarythreads, or the like. In other words, the cannula 208, without theinclusion of the fitting (not shown), may be connected directly to thepump block 402.

The at least one movable pump component is the diaphragm 404 whichdeforms into the pump block 402 to occupy a portion of the pump volumein response to a stimulus. The stimulus may include, but is not limitedto, electrical energy, thermal energy, acoustic energy, appliedpressure, or electromagnetic energy. The diaphragm 404 may be composedof crystal, ceramic, polymers, plastics, metal, glass, or combinationsthereof.

The diaphragm 404 and the cannula 208 may be hydraulically coupled, suchthat the volume from the tube end 216 of the cannula 208 and into thepump block 402 is filled with a hydraulic fluid 226. The volume from thetube end 216 of the cannula 208 and into the pump block 402 filled withthe hydraulic fluid 226, which may also be referred to as a pump volume,may be contained in a substantially rigid structure, with the exceptionof the diaphragm 404, rather than a flexible structure, to maintain thehydraulic coupling efficiency. The hydraulic fluid 226 may beincompressible or have low compressibility. The hydraulic fluid 226 maybe a solution, an oil, a liquid metal, a buffer, water, or the like.Hydraulically coupling the diaphragm 404 and the cannula 208 providesbetter small volume control (i.e. full piston travel draws/expels 1-50μL) than a non-hydraulically coupled picker (i.e. filled with air). Thehydraulic fluid 226 may include a fluid plug, such that two volumes ofthe hydraulic fluid 226 are separated by a volume of air or a differentliquid. The pump volume, as seen in FIG. 4B, which is constant,satisfies the condition given by:

V _(P) =V _(HF) +V _(MPC) +V _(PM) +V _(Air),

where V_(P) represents the pump volume, V_(HF) represents the volume ofthe hydraulic fluid, V_(MPC) represents the volume of the portion of theat least one movable pump component within the pump volume, where V_(PM)represents the volume of any picked or aspirated material, and whereV_(Air) represents the volume of air within the pump volume. In thisinstance, the at least one moveable pump component is the diaphragm 404.

FIG. 4C shows the picker 400 with the diaphragm 404 having been driveninto the pump block 402. The initial deformation distance (d_(i)) of thediaphragm 404 within the pump block 402 may be used to calculate the newvolume occupied by the diaphragm 404 within the pump volume based on theshape of the diaphragm 404. The change in deformation (Δd) from theinitial deformation distance to a second deformation distance (d_(s);where Δd=d_(s)−d_(i)) may be positive (diaphragm 404 deforms into thepump block 402; and, accordingly, creates a positive pressure gradient)or negative (diaphragm 404 withdraws from the pump block 402; and,accordingly, creates a negative pressure gradient). When the pump volumeis constant, the diaphragm 404 does not deform away from the pump block402. Therefore, the furthest that the diaphragm 404 withdraws from thepump block 402 is when the diaphragm 404 sits flush with the walls ofthe pump block 402, as seen in FIG. 4B. It should be further noted thatthe dashed line in FIG. 4C denotes the neutral state of the diaphragm404 to highlight the constant pump volume. The volume occupied by thediaphragm 404 within the pump block 402 due to the deformation of thediaphragm 404, therefore satisfies the conditions given by:

${V_{MPC} = {\frac{1}{6}{\pi \left( {d_{i} + {\Delta \; d}} \right)}\left( {{3\left( \frac{l}{2} \right)^{2}} + {3\left( {d_{i} + {\Delta \; d}} \right)^{2}}} \right)}},$

where V_(MPC) represents the at least one moveable pump component (i.e.the diaphragm 404), l represents the length of the diaphragm 404 in thepicker 400 in the neutral state (as shown in FIG. 4B and represented bythe dashed line in FIG. 4C), d_(i) represents the initial deformationdistance of the diaphragm 404 within the pump volume, and Δd representsthe change in deformation of the diaphragm 404 within the pump volume.With respect to the pump volume equation discussed above(V_(P)=V_(HF)+V_(MPC)+V_(PM)+V_(Air)), the pump volume remains constantbecause the different between the first and second volumes occupied bythe at least one moveable component (i.e. diaphragm 404) is equal to theamount of the hydraulic fluid 226, picked or aspirated material, and/orair displaced from the pump volume. For example, in FIG. 4B, d_(i) wouldbe 0, because the diaphragm 404 sits flush with the walls of the pumpblock 402. However, when the diaphragm 404 deforms, as shown in FIG. 4C,the change in deformation is Δd. Now, when the diaphragm 404 deformsagain (i.e. from FIG. 4C to another deformation), d_(i) is the initialdistance of deformation as shown in FIG. 4C and Δd is any change indeformation to the new distance of deformation.

Alternatively, when a variable pump volume is desirable, the diaphragm404 may deform away from the pump block 402. When the diaphragm 404 isable to deform away from the pump block 402, the pump volume may bevariable based on the deformation of the diaphragm.

FIG. 5 shows a cannula 500. The cannula 500 is similar to the cannula208, except that a tube end 502 of the cannula 500 includes afluorescent tip 504. The fluorescent tip 504 emits light in a particularwavelength when excited or stimulated by a stimulus, such as light witha first wavelength. The fluorescent tip 504 may be used to emit lightthat improves visualization of the cannula 500 for better placement overthe desired target analyte during collection. Alternatively, the entiretube end 502 of the cannula 500 may be composed of a fluorescentmaterial.

FIG. 6A shows a picker tip 600. FIG. 6B shows a cross-sectional view ofthe picker tip 600 taken along the line II-II. The picker tip 600 may betwo pieces, such that the picker tip 600 may be inserted into, over, orin-line with the tube end 216 of the cannula 208. Alternatively, thepicker tip 600 and the cannula 208 may be one piece. The picker tip 600includes a main body 602 and a permeable membrane 610. The main body 602includes a first end 604 with a first bore 612 having a first diameterand a second end 606 with a tapered bore 614 having a second diameterwhich tapers to the same diameter as the first diameter of the firstbore 612. The second end 606 may be entirely fluorescent or a portionthereof may be fluorescent, or the second end 606 may not befluorescent. The second diameter may be larger or smaller than the firstdiameter. Furthermore, the widest part of the tapered bore 614 may beless than or equal to 1 micrometer or less than or equal to 1millimeter.

The first end 604 is inserted within the tube end 216 of the cannula208. The permeable membrane 610 may be located within the first bore 612or the second bore 614 and is composed of a material including at leastone pore. The permeable membrane 610 permits the target analyte to bedrawn a distance into the picker. The picker tip 600 may also include aridge 608 extending circumferentially from the main body 602 to preventthe picker tip 600 from translating further into the tube end 216 of thecannula 208.

FIG. 7A shows a picker tip 700. The picker tip 700 may be inserted into,over, or in-line with the tube end 216 of the cannula 208.Alternatively, the picker tip 700 may be formed, molded, machined or thelike as a single piece with the tube end 216. The picker tip 700includes a first end 702, a second end 704, and a central bore 706. Thefirst end 702 is the portion of the picker tip 700 which be insertedinto, placed over, or placed in-line with the tube end 216 of thecannula 208. The picker tip 700 may be straight, tapered, or acombination thereof. The central bore 706 extends from the first end 702to the second end 704 and may be straight, tapered, or a combinationthereof. Furthermore, the portion of the central bore 706 at the secondend 704 may be less than or equal to 1 micrometer or less than or equalto 1 millimeter.

Magnified view 708 shows the second end 704 with an outer segmentremoved to reveal the inner configuration of the second end 704. Thesecond end 704 may be flat or angled. The second end 704 may alsoinclude a counter-sink, as shown in FIG. 7A.

FIG. 7B shows a picker tip 710. The picker tip 710 is similar to thepicker tip 700 except that the picker tip 710 includes a sharpenedsecond end 712. Magnified view 716 shows the sharpened second end 712having an outer wall 714 has an angle (θ) from the horizontal that mayrange from approximately 30° to approximately 89°. The sharpened secondend 712 permits for better cutting of the desired target analyte fromthe substrate. The angle (θ) of the outer wall 714 permits selection ofthe desired target analyte without destroying any other analytes,whether target or non-target. The central bore 706 at the second end 712may have a diameter that is less than or equal to approximately 100micrometers.

FIG. 7C shows a picker tip 720. The picker tip 720 is similar to thepicker tip 710 except that the picker tip 720 includes a flat extension722 extending from the sharpened second end 712, as seen in magnifiedview 724.

FIG. 7D shows a picker tip 730. The picker tip 730 is similar to thepicker tip 710 except that the picker tip 730 includes a flat second end732, as seen in magnified view 734.

FIG. 7E shows a picker tip 740. The picker tip 740 may be inserted into,over, or in-line with the tube end 216 of the cannula 208.Alternatively, the picker tip 740 may be formed, molded, machined or thelike as a single piece with the tube end 216. The picker tip 740includes a first end 742, a second end 744, and a central bore 746. Thefirst end 742 is the portion of the picker tip 740 which be insertedinto, placed over, or placed in-line with the tube end 216 of thecannula 208. The picker tip 740 may be straight, tapered, or acombination thereof. The central bore 746 extends from the first end 742to the second end 744 and may be straight, tapered, or a combinationthereof.

Magnified view 748 shows the second end 744 with an outer segmentremoved to reveal the inner configuration of the second end 744. Thesecond end 744 includes an opening 750 to access the central bore 746.An inner portion of the second end 744 includes a straight wall 752extending from the opening 750 into the central bore. A curved wall 754extends from the straight wall 752 further into the central bore 746. Anangled wall 758 extends from the curved wall 754 further into thecentral bore 746. An upper wall 760 extends from the angled wall furtherinto the central bore 746. In other words, the central bore 746 has afirst diameter that is substantially equal to the diameter of theopening 750, which then increases in diameter along the curved wall 754and the angled wall 758 until reaching the upper wall 760, whereby thediameter may remain constant or increase along a taper. The diameter ofthe opening 750 and the straight wall 752 may be less than or equal to 1micrometer or less than or equal to 1 millimeter. The radius of thecurved wall 754 may be approximately 25 micrometers to 2.6 millimeters.The diameter of the central bore 746 where the angled wall 758 connectsto the upper wall 760 may be approximately 125 micrometers toapproximately 2.6 millimeters.

The second end 744 may be sharpened, thereby having an outer wall 756that has an angle (θ) from the horizontal that may range fromapproximately 30° to approximately 89°. The sharpened second end 744permits for better cutting of the desired target analyte from thesubstrate. The angle (θ) of the outer wall 756 permits selection of thedesired target analyte without destroying any other analytes, whethertarget or non-target.

FIG. 8A shows picking system 800 including a drive assembly 802, thepicker 200 as shown in FIGS. 2A-2B, and an actuator 814. FIG. 8B shows across-sectional view of the picking system 800. Though picker 200 isdescribed in relation to the picking system 800, the picker 230 and thepicker 240 may also be used. The drive assembly 802 includes a driver804 including a first end and second end, a coupling 624 including afirst end and a second end, and a housing 806. The first end of thecoupling 624 mates with the second end of the driver 804, and the secondend of the coupling 624 mates with the piston 202. When the second endof the driver 804 rotates, the coupling 624 rotates, thereby causing thepiston 202 to rotate and translate within the pump block 204, thefitting 206, and the cannula 208. The driver 804, the housing 806, andthe coupling 624 translate with the piston 202 along the same axis, suchas the z-axis, relative to the pump block 204 which remains stationary.Translation of the driver 804, the housing 806, and the coupling 624translate with the piston 202 along the same axis reduces backlash, suchas by permitting use of a single-piece coupling, to allow for bettersystem control. The driver 804, the housing 806, the coupling 624, andthe piston 202 may translate the same distance along the same axis.Alternatively, the second end of the driver 804 translates along acentral axis, the coupling 624 translates along the central axis,thereby causing the piston 202 to translate within the pump block 204,the fitting 206, and the cannula 208.

The driver 804 may be an electric motor (such as a servomotor, a steppermotor, a piezo-electric actuator, a solenoid, or the like), a manualmotor (such as a knob), or the like. The driver 804 provides highresolution control of the picker 200. The coupling 624 provides zerobacklash and may be axially stiff and torsionally stiff. For example,the coupling 624 may be a single piece flexure coupling, a non-expandingbellows, split-beam drive assembly, or the like.

Furthermore, backlash may be reduced by restraining or eliminatingmovement of the driver 804 in 5 of 6 degrees of freedom. For example,the driver 804 may translate in the z-direction, but does not translatein the x- or y-directions nor does the driver 804 rotate around the x-,y-, or z-axes. Additionally, reduced or eliminated backlash permits forgreater clearance between at least the pump block 204 and the housing806, which thereby reduces the friction between the at least twocomponents, as well as better self-alignment of the piston 202 withinthe picking system 800.

The housing 806 encases and protects at least the second end of thedriver 804, the coupling 624, the first end 210 of the piston 202, andat least a portion of the pump block 204. The housing 806 may inhibitrotation of the driver 804 relative to the pump block 204. The housing806 also supports the driver 804. The housing 806 may be fixedlyattached to the driver 804. The housing 806 may include a travel slot(not shown) and a screw 810, such as a shoulder screw, to set themaximum permissible travel of the pump block 204 relative to the housing804. The screw 810 is inserted through the travel slot (not shown) andscrewed into a threaded hole on a side of the pump block 204.Alternatively, the screw 810 may be inserted through the travel slot(not shown) and compressed against a side of the pump block 204.

At least one side of the pump block 204 may be biased against at leastone side of the housing 806 to inhibit rotational motion between thepump block 204 and the housing 806 so as to reduce or eliminatebacklash. For example, a spring (not shown) may be placed between thepump block 204 and the housing 806 below the screw head of the screw810.

The housing 806, by supporting the driver 804 and only encasing aportion of the driver 804, may reduce or eliminate expansion of thepicker 200 that may result from the heat generated by the driver 804.Decoupling or separating the picker 200 and the driver 804 may reduce oreliminate expansion of the components of the picker 100. Furthermore,the weight of the driver 804 and external constraints 622, such assprings or weights, bias and preload the threads of the piston 202 toreduce or eliminate change or backlash. When the external constraints622 are springs, the springs may extend from the housing 806 to a base812. When the external constraints 622 are weights, the weights may beplaced on top of the driver 804 or the housing 806.

The drive assembly 802 may also include a home switch 808 to return thepicker 200 to the home or original position. The drive assembly 802 mayalso include a driver knob 818 for manual operation and/or wire leads620 for automated operation. Manual operation may include adjustments ormovements to the picker or picking system by hand or may includemotorized adjustments or movements to the picker or picking by anoperator via a manual controller, such as a touch screen, a joystick, adirectional pad or the like.

The picking system 800 also includes the actuator 814, such as apiezo-electric actuator, a lead screw, or a stage. The actuator 814 maybe connected to the picker 200, such as by the base 812, or may beconnected to the drive assembly 802. The base 812 supports the picker200 and may connect the actuator 814 to the picker 200. The base 812 mayinclude a light source (not shown), such as a LED, to provide epi-,transmitted, or oblique illumination of the picker tip or tube end ofthe cannula.

The actuator 814 provides high resolution location control of the picker200, has a rapid response (for example, to allow for oscillation), andmay be operated in an open or closed loop. The actuator 814 may providemotion along the x, y, and z axes or may provide motion along only oneaxis. The actuator 814 may have a travel range of 1 nanometer to morethan 50 millimeters along each axis. The lower end of the travel rangepermits the actuator 814 to make finer adjustments (approximately0.001-500 μm) for the picker 200 so as to better locate and pick atarget analyte. The upper end of the travel range permits the actuatorto make coarser adjustments (approximately 10-50 mm) for the picker 200,such as to move the picker to different wells to draw up or expeldifferent fluids from the different wells or receptacles, to changecannulas or replace parts when it is desirous to do so. The cannula orpicker tip, for example, may be replaced by manual operation (i.e.changing out by hand) or by automated operation (i.e. by expelling theused cannula or picker tip, moving the picker over a cartridgecontaining at least one new cannula or picker tip, lowering the pickerto mate with the new cannula or picker tip, raising the picker, andreturning to a desired position). When the actuator 814 provides motionalong only one axis, a second actuator (not shown) may be used toprovide motion along all three axes. Furthermore, when the actuator 814provides motion along only one axis, the second actuator (not shown) maybe used for coarser adjustments, whereas the actuator 814 may be usedfor finer adjustments.

The picking system 800 may also include a mount 816 to attach the picker200, the drive assembly 802, and the actuator 814 to an imaging ordetection system, such as a scanner or a microscope. The mount 816 maybe stationary within the imaging or detection system or may be attachedto the second actuator (not shown) within the imaging or detectionsystem.

The picking system 800 may also the port 312. The port 312 may extendthrough the base 812, the second end of the pump block 204 and eitherthe first end 218 of the fitting 206 or the adapter 214 of the cannula208. The port 312 may be rigid. The port 312 may be connected to theloader 314, such as a piezoelectric pump, to fill the pump volume. Theloader 314 may be connected to the reservoir 316 with the tube 318. Thetube 318 may be rigid or flexible. The loader 314 may be mounted on thepump block 204, such as by a screw, adhesive, tape, or the like. Thereservoir 316 may also be mounted on the pump block 204, such as by ascrew, adhesive, tape, or the like; or, the reservoir 316 may be held ormounted on or within a scanning or imaging device. Alternatively, theloader 314 may be interchanged with a pressure sensor (not shown) tomeasure the pressure within the pump volume to make sure the picker 310does not clog.

The picker can be composed of a variety of different materialsincluding, but not limited to, ceramics; glass; metals; organic orinorganic materials; plastic materials; and combinations thereof. Thepicker tip can also be composed of a variety of different materialsincluding, but not limited to, ceramics; glass; metals; organic orinorganic materials; plastic materials; polymers; jewels (i.e. ruby,sapphire, or diamond); and combinations thereof. Furthermore, thecannula or the picker tip may be composed of a material that isfluorescent. Additionally, the tube end of the cannula or the picker tipmay be impact-resistant, hard, and dimensionally stable (i.e. axiallyand/or torsionally stiff). The tube end of the cannula or the picker tipmay have a density that is greater than or equal to approximately 2.5g/cc. The tube end of the cannula or the picker tip may have a hardnessthat is greater than or equal to approximately 80 Vickers. The tube endof the cannula or the picker tip may have a Modulus of Elasticity thatis greater than or equal to approximately 65 GPa.

The permanent magnet includes, but is not limited to, a ring magnet, abar magnet, a horseshoe magnet, a donut-shaped magnet, a sphericalmagnet, a polygon-shaped magnet, a polyhedral shape, a wand magnet, akidney-shaped magnet, a trapezoidal magnet, a disk magnet, a cow magnet,a block or brick magnet, or combinations thereof. The magnetizablematerial includes, but is not limited to, metals, organic materials,inorganic materials, minerals, ferrofluids, and combinations thereof.

The cannula, picker tip and engagement portion may be stiff, flexible orformable. The cannula, picker tip and engagement portion may bestraight, angled, curved, hooked, or any appropriate shape orconfiguration. The cannula, picker tip, and engagement portion may benon-clogging.

Detecting a Picker or Picking System

A picker or picking system may be used to isolate a target analyte froma suspension in or on a vessel, such as a well, a well plate, a slide, atube, or the like, or to draw a fluid, such as a, suspension, solutionor reagent, from the vessel. For example, to isolate the target analyte,the suspension suspected of containing the target analyte can be placedin the vessel. Alternatively, a fraction of the suspension, the fractionsuspected of containing the target analyte, can be placed in the vessel.The vessel may be imaged to detect the target analyte and determine thelocation of the target analyte. After determining the location of thetarget analyte, an open end of the picker or picking system, such as thesecond end of a picking tip, the tube end of a cannula, or a tip, isguided to be located above the desired target analyte. The open end maythen be moved toward the vessel until the open end eventually touchesthe vessel or is submerged within the suspension.

To properly determine the location of the open end, the vessel andpicker or picking system may be imaged while guiding the open end intothe proper position. During this imaging, the picking tip, cannula, ortip may oscillate back and forth. However, the picking tip, cannula, ortip may stop oscillating when in contact with the vessel or suspension.Therefore, by noting the point at which the oscillating terminates, thelocation of the picking surface may be determined and the open end maybe properly visualized.

Alternatively, a sensor, such as a pressure sensor or a proximitysensor, may be placed in various locations on the imaging device, suchas on a stage, in a picker or picking system component, in a turret, orthe like, to detect a change in pressure or location, thereby denotingwhen the tip, cannula, or picking tip has contacted the picking surface.

Isolating a Target Analyte with a Picker or Picking System

A picker or picking system may be used to isolate a target analyte froma suspension. The picker or picking system may be used in conjunctionwith a vessel, such as a well, a well plate, a slide, or the like. Forexample, to isolate the target analyte, the suspension suspected ofcontaining the target analyte can be placed in the vessel.Alternatively, a fraction of the suspension, the fraction suspected ofcontaining the target analyte, can be placed in the vessel. The vesselmay be imaged to detect the target analyte and determine the location ofthe target analyte. After determining the location of the targetanalyte, the target analyte may be isolated by the introduction of aforce, such as a pressure gradient, to draw the target analyte into apicker. For example, to introduce a negative pressure gradient with thepicker 200, thereby drawing the target analyte into the cannula orpicker tip, the piston 202 translates away from the vessel. To introducea positive pressure gradient with the picker 200, thereby expelling thetarget analyte or a releasing fluid, the piston 202 translates towardsthe vessel.

To remove the target analyte from a wet mount or a suspension, anegative pressure gradient may be introduced by the picker 200 after thecannula 208 is placed near, over, or above the target analyte. Thenegative pressure gradient causes the target analyte to move into thecannula 208.

To remove the target analyte from a dry mount (i.e. a dry slide), thecannula 208 is placed over the target analyte. The cannula 208 may thenbe moved horizontally or orthogonally to detach the target analyte fromthe mount. A negative pressure gradient may then be introduced to drawthe target analyte into the cannula 208. Alternatively, the cannula 208,after being placed over the target analyte, may oscillate up and down atany appropriate frequency to detach the target analyte from the mount,such as, for example, less than or equal to approximately 10 kHz. Anegative pressure gradient may then be introduced to draw the targetanalyte into the cannula 208. Alternatively, the cannula 208 may beplaced over the target analyte and the target analyte may be held withinthe cannula without actively applying the pressure gradient.Alternatively, the cannula 208 may be placed over the target analyte anddragged across the surface of the slide, thereby dislodging the targetanalyte and causing the target analyte to be held within the cannulawithout actively applying the pressure gradient. Alternatively, areleasing fluid may be expelled by the cannula or picker tip byintroducing a positive pressure within the picker. The releasing fluid,such as a detergent, a lysing agent, a permeabilizing agent, phosphatebuffered saline, or the like, may be added on top of the desired targetanalyte so as to release the target analyte from the dry mount. Anegative pressure gradient may then be introduced to draw the targetanalyte into the cannula or picker tip.

To expel the target analyte or fluid from the picker, a positivepressure gradient may be introduced by the pump. Additionally, the tubeend of the cannula or the picker tip may be moved to touch the surfaceof the substrate onto which the target analyte or fluid is beingdispense and then moved away from the surface to wick the target analyteor fluid onto the surface.

The target analyte may be collected, and once collected, the targetanalyte may be analyzed using any appropriate analysis method ortechnique, though more specifically intracellular analysis includingintracellular or extracellular protein labeling; nucleic acid analysis,including, but not limited to, protein or nucleic acid microarrays;FISH; or bDNA analysis. These techniques require isolation,permeabilization, and fixation of the target analyte prior to analysis.Some of the intracellular proteins which may be labeled include, but arenot limited to, cytokeratin (“CK”), actin, Arp2/3, coronin, dystrophin,FtsZ, myosin, spectrin, tubulin, collagen, cathepsin D, ALDH, PBGD,Akt1, Akt2, c-myc, caspases, survivin, p27^(kip), FOXC2, BRAF,Phospho-Akt1 and 2, Phospho-Erk1/2, Erk1/2, P38 MAPK, Vimentin, ER, PgR,PI3K, pFAK, KRAS, ALKH1, Twist1, Snail1, ZEB1, Slug, Ki-67, M30, MAGEA3,phosphorylated receptor kinases, modified histones, chromatin-associatedproteins, and MAGE. In order to fix, permeabilize, or label, fixingagents (such as formaldehyde, formalin, methanol, acetone,paraformaldehyde, or glutaraldehyde), detergents (such as saponin,polyoxyethylene, digitonin, octyl β-glucoside, octyl β-thioglucoside,1-S-octyl-β-D-thioglucopyranoside, polysorbate-20, CHAPS, CHAPSO,(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol or octylphenolethylene oxide), or labeling agents (such as fluorescently-labeledantibodies, Pap stain, Giemsa stain, or hematoxylin and eosin stain) maybe used.

It should be understood that the method and system described anddiscussed herein may be used with any appropriate suspension orbiological sample, such as blood, bone marrow, cystic fluid, ascitesfluid, stool, semen, cerebrospinal fluid, nipple aspirate fluid, saliva,amniotic fluid, vaginal secretions, mucus membrane secretions, aqueoushumor, vitreous humor, vomit, and any other physiological fluid orsemi-solid. It should also be understood that a target analyte can be acell, such as ova or a circulating tumor cell (“CTC”), a fetal cell(i.e. a trophoblast, a nucleated red blood cell, a fetal white bloodcell, a fetal red blood cell, etc.), a circulating endothelial cell, animmune cell (i.e. naïve or memory B cells or naïve or memory T cells), avesicle, a liposome, a protein, a nucleic acid, a biological molecule, anaturally occurring or artificially prepared microscopic unit having anenclosed membrane, a parasite, a microorganism, or an inflammatory cell.

The foregoing description, for purposes of explanation, used specificnomenclature to provide a thorough understanding of the disclosure.However, it will be apparent to one skilled in the art that the specificdetails are not required in order to practice the systems and methodsdescribed herein. The foregoing descriptions of specific embodiments arepresented by way of examples for purposes of illustration anddescription. They are not intended to be exhaustive of or to limit thisdisclosure to the precise forms described. Many modifications andvariations are possible in view of the above teachings. The embodimentsare shown and described in order to best explain the principles of thisdisclosure and practical applications, to thereby enable others skilledin the art to best utilize this disclosure and various embodiments withvarious modifications as are suited to the particular use contemplated.It is intended that the scope of this disclosure be defined by thefollowing claims and their equivalents:

I/We claim:
 1. A tip comprising: a first end; a second end including anouter wall having an angle from the horizontal of approximately 30° toapproximately 89°; and a central bore extending from the first end tothe second end, wherein the second end includes an opening to access thecentral bore and a straight wall extending from the opening into thecentral bore, and wherein the straight wall and the outer wall arejoined directly to one another.
 2. The tip of claim 1, wherein thesecond end includes a curved wall extending from the straight wallfurther into the central bore, an angled wall extending from the curvedwall further into the central bore, and an upper wall extending from theangled wall further into the central bore
 3. The tip of claim 2, whereinthe diameter of the opening and the straight is less than or equal to 1micrometer or less than or equal to 1 millimeter.
 4. The tip of claim 3,wherein the radius of the curved wall is approximately 25 micrometers to2.6 millimeters.
 5. The tip of claim 4, wherein the diameter of thecentral bore where the angled wall connects to the upper wall isapproximately 125 micrometers to approximately 2.6 millimeters.
 6. Thetip of claim 5, wherein central bore maintains a constant diameter alongthe upper wall.
 7. The tip of claim 5, wherein the central bore has anincreasing diameter along the upper wall.
 8. The tip of claim 2, whereinthe radius of the curved wall is approximately 25 micrometers to 2.6millimeters.
 9. The tip of claim 2, wherein the diameter of the centralbore where the angled wall connects to the upper wall is approximately125 micrometers to approximately 2.6 millimeters.
 10. The tip of claim2, wherein central bore maintains a constant diameter along the upperwall.
 11. The tip of claim 2, wherein the central bore has an increasingdiameter along the upper wall.
 12. The tip of claim 1, wherein at leastthe second end is composed of at least one of a ceramic, glass, a metal,an organic material, an inorganic material, a plastic material, apolymer, or a jewel.
 13. The tip of claim 1, wherein at least the secondend has a density that is greater than or equal to approximately 2.5Wee.
 14. The tip of claim 13, wherein at least the second end has ahardness that is greater than or equal to approximately 80 Vickers. 15.The tip of claim 14, wherein at least the second end has a Modulus ofElasticity that is greater than or equal to approximately 65 GPa. 16.The tip of claim 1, wherein at least the second end has a hardness thatis greater than or equal to approximately 80 Vickers.
 17. The tip ofclaim 1, wherein at least the second end has a Modulus of Elasticitythat is greater than or equal to approximately 65 GPa.
 18. The tip ofclaim 1, wherein the angle of the outer wall from the horizontal isapproximately 30° to approximately 80°.
 19. The tip of claim 19, whereinthe angle of the outer wall from the horizontal is approximately 30° toapproximately 60°.
 20. The tip of claim 1, further comprising apermeable membrane within the central bore.